Glycan analysis is increasingly applied in biological research, clinical analysis, and pharmaceutical/biotechnological production methods. Specific glycosylation patterns have been associated with states of health and disease. (Peracaula R, et al. (2003) Glycobiology 13:457-470; Saldova R et al., (2007) Glycobiology 17:1344-1356).
Saccharides play essential biological function, for instance, O-, N-glycans in glycoproteins, O-/M-antigens in lipopolysaccharides (LPS). However, saccharides are difficult to separate and measure in nature due to lack of chromophores and low ionization ability in mass spectrometry (MS). Thus, saccharides are undetectable by conventional photometric methods (e.g. UV/fluorescence in liquid chromatography) or MS analysis. (Price N P J et al., Anal. Chem. 2010, 82, 2893; Pabst M. et al., Anal. Biochem. 2009, 384, 263; Tripathi, R. P., et al. Current Org. Chem. 2008, 12, 1093; Bigge J. C., et al., Anal. Biochem. 1995, 230, 229; Harvey, D. J. J. Chromatogr. B 2011, 879, 1196; Hase S. et al., J. Biochem. 1984, 95:197)
Saccharide labeling is an important topic in analytical chemistry. (Ruhaak L R et al., Anal Bioanal. Chem. 397:3457-3481 (2010)). Although some methods have been developed for saccharide labeling, these approaches suffer from some drawbacks. (Lin, C et al., J. Org. Chem. 2008, 73, 3848-3853; Lin, C et al., Rapid Commun Mass Spectrom. 2010, 24, 85-94; Lin, C et al., Molecules 2010, 15, 1340-1353; Lin, C et al., Molecules 2011, 16, 652-664; Chang, Y. L et al., J. Mass Spectrom. 2011, 46, 247-255; Kuo C-Y et al., Molecules 2011, 16, 1682-1694). An example is the tagging of free saccharides by reductive amination with amino-phospholipids or with neutral or acidic fluorescent reagents. (Hase S. et al., J. Biochem. 95:197-203 (1996)). Although this approach is useful and practical in some applications, fractionation of saccharides tagged by these methods has certain limitations.
Conjugation (covalent coupling) of polysaccharides to proteins or peptides has been used previously. Coupling polysaccharide fragments to proteins or peptides through their reducing end by direct reductive amination leads to poor incorporation of the saccharides.
Although there are some successful methods in saccharide labeling as discussed above, these previously reported methods still have some limitations. For example, using these previously reported methods, O-/N-glycans, which are novel and tiny from cellular source, do not reverse for biological assay after reductive amination or C-glycosylation labeling. Actually, the tagged saccharides might have different biological effects from native saccharides. In addition, the labeling reactions through N-condensation (ex. oximes and hydrazones) are reversible process between both tagged and untagged saccharides in acidic conditions (FIG. 1). This physical phenomenon makes them unstable and results in incomplete tagging during reaction and storage.
Thus, there is a long-felt need for a stable, removable tag in glycan chemistry.